Epiphytic bacterial communities on the surfaces of marine algae are poorly characterised. Most information available on marine bacterial epiphytes is derived from culture-based studies. With the rapid development of molecular community analysis technologies, it is now possible to obtain a more comprehensive picture of marine microbial populations on living surfaces. The intertidal macroalga Ulva australis, belongs to the cosmopolitan group of green marine algae (Ulvales) known to require the presence of bacteria for normal growth and has been suggested to employ specific bacteria for the defence against fouling by micro- and macro-organisms. This thesis has examined the composition and structure of the surface associated bacterial community on Ulva australis using 16S rRNA gene clone library, denaturing gradient gel electrophoresis (DGGE), and catalysed reporter deposition ??? fluorescence in situ hybridisation (CARD-FISH) analysis. The 16S rRNA gene clone library revealed that the five main bacterial groups present in the surface associated community were Bacteriodetes, Planctomycetes, Alpha-, Gamma-, and Delta-Proteobacteria. Twenty-two sequence phylotypes were identified, suggesting that the epiphytic community was of relatively low diversity. A clone similar to an algal morphogenesis inducing Cytophaga strain was identified, indicating that U. australis harbours bacteria important for thallus structural maintenance. DGGE analysis showed that while the bacterial community varied over spatial and temporal (seasons) scales it also included a stable subpopulation consistently associated with the seaweed surface. Sequencing of selected DGGE bands suggested that members of the Alphaproteobacteria and the Bacteriodetes belonged to the stable subpopulation. Using CARD-FISH with different phylogenetic probes demonstrated that Alphaproteobacteria (~ 70%) and Cytophaga-Flavobacteria (~13%) constituted the majority of bacterial cells on the surface of U. australis. A comparison of the results provided by the molecular community analysis methods, employed in this thesis, and those of culturing of epiphytic bacteria from U. australis revealed that each approach provides different patterns of phylogeny and extent of diversity. For example, the culture collection and the clone library detected a relatively high amount of Gammaproteobacteria, however, DGGE and CARD-FISH did not. Also, low species diversity clone sequences and isolates of Alphaproteobacteria contrasted with the high numbers detected by the DGGE analysis. In addition to the phylogentic determination of the epiphytic bacterial community, CARDFISH was also used to assess the organisation and distribution of bacterial cells across different zonal regions on seaweed surface. It was found that approximately 40% of bacterial cells clustered in aggregates, or microcolonies. These aggregations were considered to be heterogeneous in composition and were mainly comprised of multiply species. The occurrence of more non-viable solitary single rather than aggregated cells suggests that aggregates might offer greater protection to bacterial cells from the harsh conditions in the intertidal zone. More broadly, CARD-FISH was found to be a useful tool for studying microcolonies and was also successfully applied to detect slow growing soil microcolonies cultivated using a novel soil substrate membrane system culturing technique without the need to perform an rRNA enrichment incubation. The findings in this thesis, as described from the application of a number of molecular community analysis techniques such as clone library, DGGE and CARD-FISH, have improved our understanding of the diversity and structure of the epiphytic bacterial community associated with U. australis. Morevover, the information provided may to design future studies in the ecology of bacteria-seaweed interactions, including symbiotic interactions, and aid in marine biotechnology applications such as identifying bacteria which produce bioactive secondary metabolites.
| Identifer | oai:union.ndltd.org:ADTP/257154 |
| Date | January 2006 |
| Creators | Tujula, Niina Amanda, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Niina Amanda Tujula, http://unsworks.unsw.edu.au/copyright |
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